After the National Human Genome Research Institute and Celera Genomics have competitively researched and studied human genome under the Human Genome Project, the information map of human gene was completed in 2000, which put spurs to the development of a diagnostic oligo-DNA chip using studies on DNA functions, variant DNA search, and the information thereon. In particular, the oligo-DNA chip for searching a single nucleotide polymorphism (SNP) is expected to be used for diagnosing genetic diseases caused by defective DNA, or cancer at an early stage, etc. Further, it has been a world-wide trend to research and study the technology for manufacturing an oligo-DNA chip wherein a number f oligo-DNAs are fixed on a substrate, the oligo-DNA chip providing information on accurate virus infection routes based on the virus typing, analysis on its harmfulness or harmlessness, etc., and the prediction of occurrence of a cancer, etc. based on the typing of a group of carcinogenic viruses. In fact, an oligo-DNA chip that can type the human papilloma virus (HPV) has been officially approved by the Korea Food & Drug Administration (KFDA) in Korea for the first time in the world as a diagnostic and preventive medical biochip for predicting the probability of the occurrence of cervical cancer. Further, the oligo-DNA chip for diagnosing the cervical cancer has already been officially approved by KFDA, and it is now in use.
An oligo-DNA chip comprises short DNAs (oligo-DNAs) being 15-50mer fixed on a substrate. Thus, whereas the c-DNA chip, which has been used for analyzing DNA variants, conventionally utilized a simple physical absorption method, the oligo-DNA chip still utilizes the method that was developed several decades ago, such as a fixing method based on the use of the imine bonding method by a chemical bonding between aldehyde and amine; or a fixing method based on the amine bonding method following a reduction reaction to increase the bonding after the chemical bonding between aldehyde and amine functional group. It is because it has not yet been successful to develop a bio-chip substrate that is superior to the aldehyde-chip in terms of reproducibility and convenience. However, in the aldehyde-chip substrate, the amine functional group attached to the terminal of the oligo-DNA by means of a synthesis technology is chemically bonded by an imine bonding onto the aldehyde-chip; thus, there is a huge difference in the theoretical maximum density between the amount of the fixed DNA and the amount of the fixed oligo-DNA that is combinable with c-DNA in an actual hybridization reaction. Further, the density of the fixed oligo-DNA constantly changes because it is heavily influenced by the fixation conditions, and thus the development of a chip with reproducibility has not been successful.
A technology applying the bonding method of streptavidin-biotin is also widely used, and said technology is a method for producing oligo-DNAs by fixing streptavidin-biotin onto the surface of a solid substrate by means of a physical absorption, chemical bonding, etc., and then by making streptavidin-biotin molecularly recognize the biotin functional group of the oligo-DNA to which the biotin is attached (Science, 1993; Vol. 262, pp 1706-1708). However, the technology applying the chemical bonding method or the bonding method of streptavidin-biotin, etc. is still behind the aldehyde-chip substrata in performance.
Meanwhile, in a protein chip, the active position of an antibody exists at one place of the protein fixed on the surface, and thus there would be no problem in obtaining results if only said place is located where it can bond with an antigen. However, in an oligo-DNA chip, at least a certain number of bases of most of the oligo-DNAs must bond with the approaching c-DNAs in a hybridization reaction, and thus the number of the bases that bond must be maximized by getting the c DNAs as close to the floor on which oligo-DNAs are fixed as possible. To this end, an opinion has been suggested since European society of 2003 that between the fixed oligo-DNAs, there should be maintained some room to allow the c-DNAs free access, and now, a study of the technology for securing such room has become a hot subject in the art. Oligo-DNAs that are fixed on the surface at molecular level tend to voluntarily assemble together, and thus it is relatively difficult to apply the technology to the chemically bonded oligo-DNA chip. Accordingly, an oligo-DNA, 20-25-mer of which would otherwise be enough, is lengthened up to 50-mer so that the approaching c-DNA may not have to come down to the bottom, and the number of bases that bond during the hybridization reaction is increased to reproduce the result of a diagnosis. However, it takes from at least several hours to more than a day for the result to become clearly readable, and thus the technology is not distinguished from the conventional diagnostic technology concerning an analysis of Bird flue virus, etc.